Structure and operation of the

DNA-translocating type I DNA

restriction enzymes
Christopher K. Kennaway,1,8,9 James E. Taylor,2,8 Chun Feng Song,1,10 Wojciech Potrzebowski,3
William Nicholson,1,11 John H. White,4 Anna Swiderska,2 Agnieszka Obarska-Kosinska,3,12
Philip Callow,5 Laurie P. Cooper,4 Gareth A. Roberts,4 Jean-Baptiste Artero,5,6 Janusz M. Bujnicki,3,7
John Trinick,1 G. Geoff Kneale,2 and David T.F. Dryden4,13
1
Astbury Centre, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom; 2Biophysics
Laboratories, Institute of Biomedical and Biomolecular Sciences, School of Biological Sciences, University of Portsmouth,
Portsmouth PO1 2DY, United Kingdom; 3Laboratory of Bioinformatics and Protein Engineering, International Institute of
Molecular and Cell Biology in Warsaw, PL-02-109 Warsaw, Poland; 4EaStCHEM School of Chemistry, University of Edinburgh,
Edinburgh EH9 3JJ, United Kingdom; 5Partnership for Structural Biology, Institut Laue-Langevin, Grenoble, Cedex 9, France;
6
EPSAM and ISTM, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom; 7Bioinformatics Laboratory, Institute of
Molecular Biology and Biotechnology, Adam Mickiewicz University, PL-61-614 Poznan, Poland

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial
species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the
influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific
methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is
preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the
structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle
scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the
open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by
using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM
enzymes and type II RM enzymes.
[Keywords: DNA restriction enzymes; DNA translocation; electron microscopy; small-angle scattering; molecular
modeling; DNA translocases]
Supplemental material is available for this article.
Received September 14, 2011; revised version accepted November 14, 2011.

An important goal in understanding large molecular

machines is determination of their molecular architecture,
as this provides the foundation for understanding their
mechanism. It is >40 years since the ;440-kDa multifunctional type I DNA restriction/modification (RM) enzymes were first purified (Linn and Arber 1968; Meselson
and Yuan 1968). Their discovery, and particularly their
ability to cut dsDNA, led researchers onto the track of the
8

These authors contributed equally to this work.

Present addresses: 9Institute of Structural and Molecular Biology, DarwinSwann Building, The Kings Buildings, University of Edinburgh, Edinburgh
EH9 3JR, UK; 10Electron Microscopy Center, Hebei Medical University,
Shijiazhuang, Hebei 050017, China; 11Department of Biochemistry, University of Oxford, New Biochemistry Building, Oxford OX1 3QU, UK;
12
Biocomputing group, Department of Biochemical Sciences, Sapienza
University, P. le A. Moro 5, 00185 Rome, Italy.
13
Corresponding author.
E-mail david.dryden@ed.ac.uk.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.179085.111.

92

site-specific type II restriction endonucleases and helped to

usher in the era of genetic engineering (Loenen 2003).
These type I RM enzymes are found in over half of
bacterial genomes, including many pathogens (Roberts et al.
2010), and play a role in controlling horizontal gene transfer
(HGT) (Murray 2002; Waldron and Lindsay 2006). This is of
special importance in the spread of antibiotic resistance
in pathogens, such as multidrug-resistant Staphylococcus
aureus (MRSA), where the Sau1 type I RM system appears
to regulate HGT (Waldron and Lindsay 2006). The barrier to
HGT provided by the numerous variants of Sau1 appears to
define the clonal structure of S. aureus populations around
the world.
So important is the apparent role of type I RM enzymes
as a barrier to HGT that mobile genetic elements, including
phage and conjugative transposons and plasmids, have
developed an extensive range of anti-restriction measures
to overcome the RM barrier (Tock and Dryden 2005).

GENES & DEVELOPMENT 26:92104 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org

Structure of type I restriction enzymes

Anti-restriction measures directed against type II RM

systems are relatively simple, involving a reduction in
the number of target sequences on their mobile genome
or the incorporation of modified bases. Anti-restriction
measures against the type I RM systems are much more
diverse and sophisticated and include the production of
a range of specialized anti-restriction proteins and enzymes, including mimics of DNA structure (Walkinshaw
et al. 2002; McMahon et al. 2009). These DNA mimics
are highly charged and elongated proteins capable of
binding more tightly to the type I RM enzyme than even
its DNA target sequence.
In order to achieve a better understanding of fundamental aspects of HGT, further knowledge of the structure and mechanism of type I RM systems is essential. In
contrast to the situation with type II restriction enzymes,
it is only very recently that partial atomic structures for
type I RM subunits have emerged (Calisto et al. 2005;
Kim et al. 2005; Obarska et al. 2006; Obarska-Kosinska
et al. 2008; Kennaway et al. 2009; Lapkouski et al. 2009;
Uyen et al. 2009; Taylor et al. 2010; Gao et al. 2011), and
their mechanisms, which most dramatically involve the
translocation of thousands of base pairs of DNA with the
formation of supercoiled loops (Yuan et al.1980; Endlich
and Linn 1985; Studier and Bandyopadhyay 1988; Garca
and Molineux 1999), still present many questions. The
type I RM enzymes are complex structures with two HsdR
restriction (R) subunits, two HsdM modification (M) subunits, and one HsdS sequence specificity (S) subunit (Murray
2000; Loenen 2003), each with a number of domains
(Supplemental Fig. S1).
The HsdS contain two DNA target recognition domains (TRDs), one for each part of the bipartite recognition sequence, joined by two a helices in an antiparallel
arrangement. The HsdS structure displays pseudo twofold symmetry (Calisto et al. 2005; Kennaway et al. 2009;
Kim et al. 2005; Gao et al. 2011), and this symmetry is
imposed on the whole structure of these enzymes (Kneale
1994; Dryden et al. 1995; Davies et al. 1999; Kennaway et al.
2009). The two HsdM lie on either side of the HsdS to form
the M2S1 methyltransferase (MTase) core. The HsdM contain a methyltransferase catalytic domain. The two HsdR
are located on either side of the MTase core and contain a
nuclease domain and an ATP-hydrolyzing DNA translocation motor domain (Powell et al. 1998; Davies et al. 1999).
The MTase methylates specific adenine bases in the
target sequences on hemimethylated, newly replicated
host DNA (Dryden 1999; Madhusoodanan and Rao 2010),
while the HsdR are switched on only when unmodified
target sequences are detected on incoming DNA. Although
unmodified targets can appear on host DNA during times of
cell stress, restriction of host DNA is prevented by restriction alleviation (Makovets et al. 2004). The restriction
reaction appears to work by fixing the enzyme to the
recognition sequence and initiating an extended period of
ATPase-driven DNA translocation (Seidel et al. 2008),
which pulls DNA in toward the enzyme from both sides
of the target site. Loops of DNA are extruded, and when
the translocation stalls, the DNA is cleaved at the stall
site rather than the target sequence. As the enzyme tracks

the DNA helix, supercoiling can build up in the extruded

loops. How the enzyme can translocate for up to 50,000 base
pairs (bp) (Garca and Molineux 1999) and at speeds of up to
1000 bp per second (Seidel et al. 2004) in the face of the
torsional stress induced by this supercoiling is not known,
although it seems that the enzyme makes many abortive
attempts to initiate the translocation (McClelland et al.
2005).
We now present a structure of EcoKI (specificity sequence target AACNNNNNNGTGC), the archetypal
type I RM enzyme found on the chromosome of Escherichia coli K12, and EcoR124I (specificity sequence target
GAANNNNNNRTCG), found on a conjugative plasmid,
as determined by reconstruction from electron microscopy
(EM) and single-particle analysis together with small-angle
X-ray scattering (SAXS) and small-angle neutron scattering
(SANS). The structures show EcoKI bound to a DNA duplex
or to a DNA mimic anti-restriction protein, and EcoR124I
in the absence and presence of a DNA duplex or an antirestriction protein. With the aid of numerous biochemical
constraints, known crystallographic structures of type I RM
subunits, the location of the HsdS and HsdM within the
MTase at low resolution (Callow et al. 2007; Taylor et al.
2010), and our recent reconstruction of the core MTase
structure (Kennaway et al. 2009), we can construct a unique
arrangement of the subunits within the EM structures,
rationalize all previous knowledge about these complex
machines, and reveal an evolutionary link between type I
and type II RM systems.
Results
We analyzed here a variety of complexes with EM, SANS,
and SAXS; namely, EcoR124I with bound DNA (30 bp),
EcoR124I without DNA, EcoKI with bound DNA (75 bp),
and EcoR124I with bound Ocr anti-restriction protein. It
is important to note that, in the absence of ATP hydrolysis,
EcoKI is a stable assembly containing all five protein
subunits (Dryden et al. 1997; Roberts et al. 2011). However,
for EcoR124I, the binding constants for each of the two
HsdR differ by over two orders of magnitude (0.6 nM and
200 nM, respectively), and the two forms of the enzyme,
R1M2S1 and R2M2S1, are in dynamic equilibrium (Supplemental Material; Supplemental Fig. S3; Janscak et al. 1998;
Mernagh et al. 1998). The R1M2S1 form is active in translocation but not DNA cleavage (Janscak et al. 1998). At the
micromolar concentrations used for the SAXS and SANS
experiments, dissociation of the R2M2S1 complex is minimal, both with and without bound DNA; however, at the
;100 nM concentrations used for EM, there will be a considerable fraction of EcoR124I in the R1M2S1 form. In all
experiments using 1:1 protein:DNA complexes, the concentrations are above the binding constants for DNA; hence,
free enzyme and free DNA concentrations will be very low.
Negative stain EM of EcoR124I
Single-particle analysis of negative stain EM images
showed large differences between DNA-bound (Fig. 1A;
Supplemental Fig. S2A,B) and unbound EcoR124I enzymes

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Kennaway et al.

Figure 1. Gallery of type I RM structures and

conformations determined by EM and single-particle analysis. (A) EcoR124I+DNA (closed state)
negative stain EM. (B) EcoR124I without DNA
(open state) negative stain EM. (C) EcoKI+DNA
negative stain EM. For each 3 3 3 panel, the top
rows are image averages, the middle rows are their
corresponding reprojections, and the bottom rows
are 3D surface views of the 3D reconstruction
); on the right is a larger 3D surface
(bars, 200 A
perspective view. Supporting EM data can be
found in Supplemental Figure S2. Data on the
assembly of the EcoR124I enzyme can be found
in Supplemental Figure S3.

(Fig. 1B; Supplemental Fig. 2C,D), with their longest dimensions being ;18 nm versus ;2226 nm, respectively.
(Smaller particles were the R1M2S1 form and were analyzed
separately as described later.) We refer here to the form of
EcoR124I with DNA as the closed form and the form
without DNA as the open form, following the nomenclature used for the two states of the EcoR124I MTase (with
and without bound DNA) on the basis of SAXS analysis
(Taylor et al. 1994). Apparent twofold symmetry was visible
in many image averages.
A three-dimensional (3D) reconstruction was generated
(Fig. 1A; Supplemental Fig. S2A,B) of the EcoR124I RM

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GENES & DEVELOPMENT

enzyme bound to a 30-bp dsDNA fragment containing its

unmethylated recognition site (Supplemental Material; Supplemental Fig. S3). This map has a resolution of ;2.1 nm (by
0.5 Fourier shell correlation [FSC]) and is of good overall
quality, due to it having a reasonable range of orientations
on the grid (although still mostly limited to a single axis of
rotation) and little or no flexibility. Twofold rotational
symmetry was imposed during the refinement due to the
apparent symmetry seen in the two-dimensional (2D)
analysis and the twofold pseudosymmetry of the assembly imposed by previous experimental and theoretical
work using sequence analysis (Kneale 1994; Dryden et al.

Structure of type I restriction enzymes

1995), domain-swapping experiments (Fuller-Pace et al.

1984; Gann et al. 1987; Cowan et al. 1989; Abadjieva et al.
1993; Meister et al. 1993), and three crystal structures of
HsdS from different species (Calisto et al. 2005; Kim et al.
2005; Gao et al. 2011).
The DNA was not visible in our negative stain images.
The same problem has recently been observed in images
of the p53 protein in complex with DNA (Melero et al.
2011). This lack of DNA staining is often the case at stain
depths typical for protein staining, as it is thought that
the heavy metals bind in the DNA grooves, matching the
negative contrast (Griffith 1978). However, sometimes
the DNA is visible (Mayanagi et al. 2011), so the absence
or presence seems to depend on the particular system
investigated.
EcoR124I without DNA in negative stain was highly
extended and more flexible with a very limited range of
orientations on the carbon film (Fig. 1B; Supplemental
Fig. S2C,D). Most particles (;80%) appeared to have their
twofold axis roughly normal to the plane of the carbon
film. A low-resolution (;3.5-nm) 3D reconstruction of the
EcoR124I RM complex without DNA was generated. The
angular range was limited due to the shape of the complex,
and this limits the resolution of the open conformation.
Very thin connections between the domains could be seen
in some negative stain images, but these are not well
resolved in the subsequent 3D map due to the low resolution. These thin linkers are likely pivot points for flexing to
allow the enzyme to close up. A very small proportion of
these particles (<5%) were seen to be folded up into the
closed state, indicating a dynamic equilibrium between
states in the absence of cognate DNA.
Comparing unbound open EcoR124I with the DNAbound closed state shows a very large movement of the
subunits within the assembly (Fig. 1A,B). It is possible that
the carbon film affects the unbound open state and that it
has a more variable conformation in solution. However,
the twofold symmetry seen in many of the particles suggests that this open conformation is relatively stable, given
that random conformational variability would break the
symmetry. Opening and closing by type I RM enzymes in
such a manner must allow entry of DNA substrate. SAXS
and SANS data previously showed that the EcoR124I
MTase collapsed from an extended structure to a more
compact form in the presence of DNA (Taylor et al. 1994,
2010), as also shown by the almost complete protection
from proteolysis when the MTase was bound to DNA
(Webb et al. 1995). The recent EM analysis of the EcoKI
MTase also suggested an opening and closing cycle for
DNA binding but of lesser magnitude than for EcoR124I
(Kennaway et al. 2009).
Negative stain EM of EcoKI
Negatively stained particles of EcoKI with DNA bound
(Fig. 1C; Supplemental Fig. S2E,F) appeared smaller than
EcoR124I with DNA (;16 nm long), and the orientations
observed on the carbon grids had little similarity to those
of EcoR124I. Once again, the DNA was not visible in
negative stain even though a larger 75-bp duplex was used.

EcoKI particles showed some variability, particularly in

peripheral regions. Approximate twofold symmetry was
apparent in some views of EcoKI, and a wider range of
views were seen. This may indicate a more rounded shape
than EcoR124I, allowing adsorption of EcoKI to the carbon
film in a variety of orientations. The 3D reconstruction of
negatively stained EcoKI with a 75-bp fragment of dsDNA
displayed a compact structure with many features similar
to EcoR124I with DNA, including recognizable density for
the five subunits in a matching arrangement, suggesting
a common architecture for type I RM enzymes. EcoKI also
adopted a compact form in absence of DNA when examined by EM (the particles appeared to be identical) and did
not appear elongated, as seen for EcoR124I without DNA
(data not shown). The dynamic equilibrium between open
and closed forms apparently favors the closed form for
EcoKI under the conditions used for EM.
Small-angle scattering experiments on EcoR124I
and EcoKI
In order to determine the subunit organization of EcoR124I
and EcoKI, SAXS and SANS were performed in the absence
of DNA. The techniques are complementary, with SANS
uniquely allowing the spatial location of subunits to be
determined by matching out one or another of the components by specific perdeuteration, together with contrast
variation using a range of H2O/D2O mixtures.
For SANS measurements, EcoR124I was prepared in
either a fully protonated state or a perdeuterated state in
which HsdR was deuterated and the MTase was protonated. The HsdR were deuterated to a level of 75%, such
that the contrast match point of 100% D2O was achieved.
SANS measurements were made in buffers containing 0%,
40%, and 100% D2O (Fig. 2A). The left panel of Figure 2A
shows the scattering data, and the right panel of Figure 2A
shows the derived pair distribution function, p(r). In 0%
for the fully protonated
D2O, a radius of gyration (Rg) of 69 A
EcoR124I was determined (Table 1). Fourier transformation
of the scattering curve into a pair distribution function [p(r)]
showed that the complex had a maximum dimension
(right panel of Fig. 2A). Similar sets of mea(Dmax) of 220 A
surements were carried out in 40% D2O for the perdeuterated complex in which the MTase core was matched out and
only the scattering due to the two HsdR was visible. The Rg
and Dmax were essentially unchanged, indicating that each
HsdR must be positioned at either end of the RM enzyme,
separated by the MTase core, consistent with DNA footprinting data (Mernagh et al. 1998; Powell et al. 1998).
Finally, scattering data were collected in 100% D2O in
which the HsdR were matched out, so only the scattering
and Dmax of
from the MTase was measured. An Rg of 58 A

190 A were determined for the MTase in situ within the

body of the EcoR124I RM enzyme. Both the shape of the
curve and values of Rg and Dmax for the MTase in situ were
very similar to both SANS and SAXS results obtained for
the pure EcoR124I MTase (Taylor et al. 1994, 2010). This
indicates that the MTase in the absence of DNA has the
same tertiary structure whether the HsdR are present
or not.

GENES & DEVELOPMENT

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Kennaway et al.

shapes of both RM enzymes in the absence of DNA are

similar. From the pair distribution function (right panel of
) was slightly larger than
Fig. 2B), the Dmax of EcoKI (230 A
), possibly due to the
that determined for EcoR124I (220 A
slightly larger overall mass of EcoKI. The Rg values of EcoKI
and 70 A
, respectively, in good
and EcoR124I were 68 A
agreement with the Rg obtained by SANS for EcoR124I.
Reconstruction of the EcoR124I structure
from scattering data
An ab initio model of the EcoR124I RM enzyme, showing
the location of HsdR and the MTase, can be constructed
from the scattering experiments by both the simulated
annealing procedure in DAMMIN (Svergun 1999) and
multiphase bead modeling using MONSA (Svergun and
Nierhaus 2000). Two phases were specifiedone for the
two HsdR, and the other for the MTasewhile taking
into account the Rg determined from the Guinier region.
An overall symmetry of P2 was specified. The fit of the
complete RM enzyme model to the 0% D2O SANS data
was excellent, with a x2 value of 0.98, while the MTase and
HsdR fitted the 100% and 40% D2O SANS with x2 values of
1.47 and 1.9, perhaps indicative of the flexible nature of the
HsdR with respect to the MTase core. The MTase component was found to be located centrally within the envelope
of the RM enzyme, while each HsdR was extended toward
the outermost edge of the complex (Fig. 2C).
Location of HsdR in EcoR124I using EM

Figure 2. SANS and SAXS analyses. (A) SANS profiles of

EcoR124I. The left panel shows the scattering data, and the
right panel shows the pair distribution functions, p(r). (Gray)
Protonated EcoR124I in 0% D2O; (blue) MTase core in situ
within the RM enzyme (deuterated HsdR and protonated MTase
measured in 100% D2O); (red) the two HsdR in situ in the RM
enzyme (deuterated HsdR and protonated MTase measured in
40% D2O). (B) SAXS profiles of EcoR124I (black) and EcoKI
(green). The panel on the left shows the scattering data, and the
right panel shows the pair distribution functions, p(r). In both A
and B, the solid lines in the scattering data represent the fits
from the corresponding back-transformed distance distribution
functions, p(r), in the panel on the right. (C) Multiphase ab initio
modeling showing the location of the MTase core (blue) and the
HsdR (red), superimposed on the EM map of EcoR124I from
Figure 1B (gray). The panel on the right shows a 90 rotation
about the long axis in the left panel. Data on the assembly of the
EcoR124I enzyme can be found in Supplemental Figure S3.

SAXS data were also collected on the EcoKI and EcoR124I

RM enzymes (Fig. 2B; Table 1). The scattering curves for
both EcoKI and EcoR124I were essentially identical within
error (left panel of Fig. 2B), indicating that the overall

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GENES & DEVELOPMENT

The SANS data clearly indicate that EcoR124I is an

elongated structure with the two HsdR located toward the
extreme ends on either side of the MTase core. The protein
concentration used in the scattering experiments is substantially greater than the binding constant for the assembly of the R2M2S1 complex. However, at the lower protein
concentration used for EM, a fraction of the enzyme will
exist as the R1M2S1 form. In negative stain EM, the particles
of EcoR124I with (Fig. 3A) or without (Fig. 3B) DNA were
not 100% homogeneous, as smaller particles were noticed
following alignment and classification. These smaller particles showed a single, strongly preferred orientation on the
carbon, while the larger particles appeared to adopt a range
of orientations. Some large particle views appeared to be in
an orientation similar to those of the smaller particles,
allowing a comparison to be made. Subtracting these similarly oriented images from each other to form difference
images unambiguously showed a large missing region at
the extremity of the small particles, which, consistent with
the SANS analysis, must be the location of one of the
HsdR. The observation of complexes with only one HsdR
is consistent with previous biochemical data on EcoR124I
(Janscak et al. 1998).
Location of the DNA path in EcoR124I using a DNA
mimic protein and determination of the orientation
of the MTase in the RM enzyme
Having located the HsdR and the MTase core, ambiguities remain concerning the orientation of these units and

Structure of type I restriction enzymes

Table 1.

SAXS and SANS data

D2O

SANS
HsdR of EcoR124I in situa
MTase core of EcoR124I in situb
EcoR124I c
SAXS
EcoR124I
EcoKI

Volume

Rg

40%
100%
0%

69.0
58.0
69.0

(2.9 3 105
3.1 3 105 A
(2.0 3 105
2.4 3 105 A
5
4.6 3 10 A (4.9 3 105

0%
0%

69.9
68.1

4.7 3 105
5.1 3 105

Mr

Dmax

)
A
)
A
)
A

226 kDa (240 kDa)

198 kDa (162 kDa)
384 kDa (402 kDa)

220
190
220

(4.9 3 105 A
)
A
(5.4 3 105 A
)
A

392 kDa (402 kDa)

420 kDa (440 kDa)

220
230

In parenthesis are the theoretical values calculated from the amino acid sequence and stoichiometry.
a
EcoR124I measured in 40% D2O with deuterated HsdR and protonated MTase.
b
EcoR124I measured in 100% D2O with deuterated HsdR and protonated MTase.
c
Fully protonated enzyme measured in H2O.

the location of DNA within the overall structure. In

particular, the lack of a clear position for the DNA would
make defining the orientation of the MTase core ambiguous within the 3D map. Fortunately, we were able to use
the Ocr DNA mimic protein, which binds very tightly to
the DNA-binding site of the type I RM enzymes (Atanasiu
et al. 2002; Walkinshaw et al. 2002), to infer the path of
DNA through EcoR124I. Unlike the DNA, Ocr should be
visible in EM experiments.
Complexes of EcoR124I with Ocr adopted a closed
conformation in EM. The presence of Ocr also unexpectedly increased the proportion of complexes containing
only one HsdR (one possibility is that the Ocr binds to
free HsdR and thus lowers the proportion of the R2M2S1
complexes). However, using only those particles large
enough to contain both HsdR, a 3D map was generated by
projection matching onto the map for the EcoR124I+DNA
complex (Fig. 4AC). 2D difference images and 3D difference maps then revealed the position of a banana-shaped
object running through the center of the RM enzyme but
tilted at an angle relative to the long axis of the 3D map

Figure 3. 2D difference images from EM data show the position

of the HsdR in the EcoR124I complex. (A) Difference imaging
between image averages of large (left) and small (right) particles
in the EcoR124I+DNA negative stain EM data set reveals a large
negative density region (red contour at 2.5 s), consistent
with a missing HsdR in the small particles. (B) Difference
imaging of HsdR in the open state of EcoR124I (without
DNA). Although the relative flexibility of the open complex
gives rise to a less well-defined difference map, a region of
negative density consistent with a missing HsdR is visible
nevertheless (red contour).

(Fig. 4D). The banana-like shape matches well with the

structure of the Ocr protein (Fig. 4E; Walkinshaw et al.
2002). This orientation of Ocr in the EM map, coupled
with the structural models of the MTase core of EcoKI
(Kennaway et al. 2009) and of EcoR124I (based on that of
EcoKI as described in the Supplemental Material; Supplemental Fig. S4A), allows only one possible orientation
of the MTase atomic models within the EM envelope (Fig.
4E). The orientation chosen for the MTase exposes the
double a-helical linker of the HsdS to the solvent. This is
consistent with previous observations on the location of
HsdS, which indicated that the linker could accommodate
both small (Gubler and Bickle 1991) and large (Kannan
et al. 1989) amino acid insertions and even a fusion with
green fluorescent protein (Chen et al. 2010) without loss of
function. Moreover, limited proteolysis showed that cleavage occurred preferentially in the a-helical region between
the two TRDs of HsdS (Webb et al. 1995), and thus the
linker must be highly accessible to solvent.
Atomic modeling of the complete RM enzymes
The above data provide some of the constraints necessary
for construction of atomic models. This procedure comprises two parts: first, completion of known crystallographic
structures of the subunits by modeling of domains not
resolved by crystallography, and second, insertion of the
complete structures into the 3D envelopes provided by
EM and scattering analyses. This latter part also benefits
from the availability of published biochemical data on
these enzymes as further constraints.
Atomic models of complete HsdR for EcoKI and EcoR124I
were constructed based on known crystal structures (Supplemental Material; Supplemental Fig. S4B). The missing C
terminus (amino acid residues 8931038) of the HsdR of
EcoR124I was modeled de novo using the program Rosetta
(Supplemental Material). Although the positions of the
central MTase core region and the HsdR are well defined,
as described above, the orientation of the HsdR in the EM
map of both EcoR124I and EcoKI was ambiguous, as the
density was rather ring-like in shape. We can, however,
propose a model that fits the data and gives the location
and directionality of the DNA motor domains by aligning
the RecA-like motor domains of the crystal structure of
the dsDNA-bound SWI2/SNF2 chromatin remodeling
translocase from Sulfolobus solfataricus (Protein Data

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Kennaway et al.

Figure 4. 2D and 3D difference mapping from EM data shows

the route of DNA/Ocr through the EcoR124I complex. (A)
Negative stain EM difference image averages of EcoR124I+DNA
and EcoR124I+Ocr from two orientations show a smaller central
area of positive difference (green contours at +4.5 s), indicating
the position of the DNA mimic Ocr, which excludes stain more
effectively than DNA. (B) Surface view of the 3D reconstruction
of EcoR124I+DNA. (C) Surface view of the 3D reconstruction of
EcoR124I+Ocr shows the central hole is mostly occluded in the
Ocr-containing complex when compared with the EcoR124I+
DNA surface shown in B. (D) Two views of the DNA/Ocr 3D
difference map (green surface, contoured at +4.5 s) overlaid onto
the EcoR124I+DNA map (gray mesh) showing the main positive
difference densities. (E) Two views of the EcoR124I+DNA 3D
map (gray mesh) with the EcoKI MTase core+Ocr atomic model
(PDB code: 2y7C) docked in as a single rigid body. (Magenta
spacefill) Ocr; (yellow ribbon) HsdS; (blue ribbon) 23 HsdM. The
path of Ocr, as predicted from the MTase structure, matches
well to the difference density.

Bank [PDB] code: 1z63) (Durr et al. 2005) with those of

HsdR (Lapkouski et al. 2009). As the direction of DNA
translocation is defined in the chromatin remodeling
translocase, it imposes a similar directionality on each
HsdR, and since these have to pull DNA in toward the
MTase core of the type I RM enzyme, the orientation of
each HsdR relative to the MTase core becomes defined.
Assuming that the DNA path between the DNA bound to
the HsdR and the DNA bound to the core MTase must
not be any longer than ;40 bp, as determined by DNA
footprinting experiments (Mernagh et al. 1998; Powell et al.
1998) and the minimum length of 45 bp of DNA required for

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GENES & DEVELOPMENT

ATP hydrolysis (Roberts et al. 2011), then the locations of

the RecA-like motor domains of the HsdR are forced to be as
shown, so that their DNA-binding sites are close to the
DNA-binding site of the MTase core. Placement of the HsdR
on either side of the MTase and interacting directly with
DNA is further supported by the length of the structure of
the ArdA anti-restriction DNA mimic protein (Nekrasov
et al. 2007; McMahon et al. 2009), which occupies the
entire DNA-binding site on type I RM enzymes. This then
allows the complete structures for the closed forms of
EcoR124I and EcoKI to be constructed as shown in Figure
5, A and B.
Placement of the HsdR forces two large kinks in the
DNA to allow the DNA to thread through the MTase core
(Fig. 5A,B). This kinked path, which effectively shortens
the through-space end-to-end distance of a duplex bound
to the RM enzyme by ;10 nm, is supported by atomic
force microscopy (AFM) measurements of complexes of
EcoR124I on DNA, which showed that binding of the
nuclease shortened the length of a long linear DNA
molecule by ;11 nm (van Noort et al. 2004). AFM
measurements of EcoKI bound to DNA also showed
a pronounced kink (Walkinshaw et al. 2002; Neaves et al.
2009), and circular dichroism analysis of EcoR124I also
indicated a large structural distortion to the DNA when
bound (Taylor et al. 1994).
A fit of subunits into the EcoKI EM density map (Fig.
5B) corresponds closely to that of the EcoR124I in the
closed state. The thin protrusions at either side of the EM
envelope for EcoKI can fit the long coiled-coil N-terminal
extensions predicted in the HsdR of EcoKI but absent in
EcoR124I (Fig. 5B; Supplemental Material; Supplemental
Figs. S1, S4B). These extensions appear to be quite variable in the EM analysis and are seen in both the presence
and absence of DNA. The function of these extensions is
unknown. Significant sequence differences exist between
the two enzymes (Supplemental Figs. S1, S4), and this
may account for other structural differences, although the
overall architecture remains unchanged.
Fitting of subunits into the lower-resolution open
EcoR124I map (Fig. 5C) was aided by the SANS data, 2D
difference imaging (Fig. 3B), and the closed atomic model
(Fig. 5A). An optimal fit was obtained by moving and
rotating each HsdMHsdR pair as a single rigid body away
from HsdS. A relatively simple ;90 rotation and an ;80
twist around a pivot point near the C terminus of HsdM
are sufficient to move between open and closed states. It
has previously been shown that HsdR and HsdM can form
a complex (Dryden et al. 1997), supporting movement of
the two subunits as a rigid body. The C-terminal residues
of the EcoKI HsdM are disordered in the crystal (PDB
code: 2ar0) (Kennaway et al. 2009) and are proteolytically
sensitive (Cooper and Dryden 1994), and so could play the
role of the flexible linker proposed here. Proteolytic removal
of this region precludes assembly of the EcoKI RM enzyme
(Powell et al. 2003). A comparison of the atomic model of
the open form of EcoR124I with that of the SAXS data using
the program CRYSOL revealed a good fit with a x2 value of
1.127. In addition, a comparison of the EM model with the
SAXS data, obtained by first filling the EM envelope with

Structure of type I restriction enzymes

Figure 5. Atomic models of EcoR124I+DNA, EcoR124I, and EcoKI+DNA docked into the EM map densities. (A) Two views of the
EcoR124I+DNA model showing the MTase core closed around DNA (green; DNA bound to each HsdR is not shown for clarity).
Adenine bases are flipped out into the active sites of each of the two HsdM (light and dark blue), induced by an ;45 bend in the DNA.
The HsdS is in yellow, and the two HsdR are shown in red, with the b sheets of the recA-like motor domains colored orange. Residues
missing from the crystal structures (the 44 and 152 C-terminal residues of HsdM and HsdR, respectively) were modeled de novo and are
shown in gray. The C-terminal regions of HsdM extend down to bind at the coiled coil of HsdS, and the HsdR C-terminal domains fill
some empty density next to the N terminus of HsdM. (B) A model for the second type I RM enzyme, EcoKI bound to DNA. Colors are
as in A, with residues modeled de novo shown in gray. The HsdS and HsdM from the MTase structure (PDB code: 2y2C) were docked in
as a single rigid body. The HsdR modeled on those from EcoR124I (PDB code: 2w00), as described in the Supplemental Material, were
placed in a position analogous to the EcoR124I model. (C) The model of EcoR124I in the open conformation (i.e., without DNA). Colors
are as in A, with residues modeled de novo shown in gray. Although the EM map is at a lower resolution, a full atomic model can be built,
aided by the EcoR124I+DNA model, SANS data, and 2D difference imaging. The HsdM and HsdR swing out as a unit away from HsdS.
The predicted hinge regions in the C termini of the HsdM (modeled in gray) and their connections to HsdS are not well resolved.

dummy atoms, showed a comparably good fit with a x2

value of 1.28. This comparison of the EM data, small-angle
scattering data, and atomic models provides confidence in
the atomic models presented.
Discussion
The data presented show how type I RM enzymes are
assembled and how they bind and distort DNA prior to the
initiation of DNA translocation driven by ATP hydrolysis.
The EM and small-angle scattering structures are of low
resolution; hence, the placement of atomic models within
the structural envelopes could be ambiguous in the absence
of data obtained using other methods. However, the type I
RM enzymes have been extensively studied biochemically,
biophysically, and genetically (Murray 2000, 2002; Loenen
2003; Tock and Dryden 2005), which provides several
further constraints on the subunit orientations and gives
confidence in the atomic models shown in Figure 5.
The structures shown in Figure 5 make it clear that there
is an equilibrium between open and closed forms of the
type I RM enzymes, with the equilibrium constant depending on the particular enzyme and the presence or absence of
DNA (and presumably the cofactors S-adenosylmethionine

and ATP, although these were not specifically examined in

this study). EcoKI prefers to be closed whether DNA is
present or not, and must therefore transiently open up to
allow DNA access to the MTase core. EcoR124I appears to
prefer an open form in the absence of DNA but is closed
with DNA bound.
It would appear possible for the type I RM enzymes to
reach the closed initiation complex with the S-shaped
DNA path (Fig. 6A) via different routes. The open form can
bind DNA nonspecifically using HsdR (left side of Fig. 6A)
and diffuse along the DNA until the MTase core recognizes
a target sequence or dissociates. The trigger for closing
and formation of the initiation complex is most likely the
recognition of the target sequence by the MTase core.
Alternatively, the closed form of the enzyme must open up
transiently to allow DNA to enter the MTase core, followed by closing of the core around the DNA (right side of
Fig. 6A) and diffusion of the enzyme on the DNA until it
either recognizes its DNA target sequence or reopens and
dissociates. Starting the process of target sequence location
and recognition via this pathway means that the motor
domains of the HsdR will have to rely on the inherent
flexibility of DNA for them to grasp it and force it into the
S-like shape shown in the initiation complex.

GENES & DEVELOPMENT

99

Kennaway et al.

Figure 6. Schematic of large-scale conformational change and

initiation of DNA looping and translocation. (A) Type I RM
enzymes exist in a dynamic equilibrium between open and
closed states (movement is shown by orange arrows, and pivot
points in C-terminal regions of HsdM are indicated by pink
dots). DNA (green) binding to form encounter complexes can
occur nonspecifically to the HsdR (red) or via the target
sequence to the MTase core (HsdM is in light and dark blue,
and HsdS is in yellow). Complete closure of the enzyme and
bending of the DNA around the HsdR produces the initiation
complex for DNA translocation. (B) The predicted complete
path of the DNA (green dots) through the atomic model of
EcoR124I with segments of bound DNA. This is the proposed
initiation complex (from Fig. 5A). During active translocation,
the DNA would then form expanding loops from each side
(light-green dots for DNA, and the direction of translocation is
shown by black arrows). The inset shows the initiation complex
turned 90 to the main panel.

The introduction of sharp bends in the DNA would

require considerable energy to be expended by the enzyme.
This may come from the transition between open and
closed forms of the RM enzyme, but it may also require the
hydrolysis of ATP by the HsdR. The models suggest that
once the enzyme has closed around DNA and the motor of
an HsdR subunit has a good grip on a segment of DNA,
further hydrolysis of ATP would push the segment bound
to the motor toward the central MTase core, as indicated by
large arrows in Figure 6. Since the MTase core is also
tightly bound to the DNA target sequence, DNA at the
bend between the segments bound to the motor and to
the MTase core would twist and perhaps even buckle,
forming the small loop shown in Figure 6B. Formation of

100

GENES & DEVELOPMENT

this highly strained loop is certain to be energetically unfavorable, in agreement with translocation measurements
for type I RM enzymes in which it appears that much ATP
is used in abortive attempts to initiate translocation (Seidel
et al. 2008). Once the loop has formed, further DNA translocation would occur as the motors pump DNA toward the
MTase core. Single-molecule experiments make it clear that
the motors can work independently (Seidel et al. 2004,
2008), perhaps explaining why early EM studies showed
both single- and double-looped structures (Yuan et al.
1980; Endlich and Linn 1985). In light of the large changes
occurring upon DNA binding, it is possible that the actively
translocating enzyme undergoes further changes in structure (e.g., in the presence of ATP). One may speculate that
this great flexibility would allow the RM enzyme to
accommodate the stresses built up during the extensive
DNA translocation periods observed for these molecular
machines. It is noteworthy, in this respect, that a process
of deassembly of the enzymes occurs after DNA cleavage,
and some of the subunitsalthough not all and depending on the particular type I RM enzymecan be reused
(Roberts et al. 2011; Simons and Szczelkun 2011).
Lapkouski et al. (2009) proposed a more speculative
atomic model of EcoR124I using their structure of HsdR,
a postulated DNA path across the subunit, and an early,
incomplete model of the MTase core (Obarska et al. 2006).
Although the current models and their model share much
in common, there are two main differences; namely, the
orientation of the HsdR with respect to the MTase core,
and the path taken by the DNA. Previously (Lapkouski
et al. 2009), the interface of HsdR with the MTase core was
not defined when compared with the models presented
here. More importantly, the DNA was proposed to bend
across the motor domains of HsdR, so that it came near to
the endonuclease domain in the same HsdR and could be
cleaved. This model would suggest that the partially assembled R1M2S1 form of EcoR124I would be able to cleave
DNA. However, no cleavage was observed for this partially assembled enzyme (Janscak et al. 1998), and thus
the Lapkouski et al. (2009) model cannot be entirely correct.
The current model suggests that the endonuclease domain
of one HsdR is in proximity to DNA translocated by the
other HsdR (Fig. 6B). This would explain the absence of
DNA cleavage by partially assembled R1M2S1 forms of
EcoR124I, despite the fact that such an assembly translocates DNA effectively (Janscak et al. 1998; Seidel et al.
2004, 2008). Thus, the current models are a significant
improvement on the previously published models (Davies
et al. 1999; Lapkouski et al. 2009).
Last, the structural models presented can be compared
with the structures of complex type II RM enzymes in
groups IIB and IIG (Roberts et al. 2003), which recognize
a target sequence but cleave at defined distances on either
side of the target (IIB) or on one side of the target (IIG).
These classes appear to contain structural domains in
common with the type I RM enzymes; namely, endonuclease domains, an HsdM-like subunit, and TRDs, but no
motor domains (Dryden 1999; Nakonieczna et al. 2009;
Shen et al. 2011). In these enzymes, the motor domains of
HsdR are missing and the endonuclease domain is directly

Structure of type I restriction enzymes

fused to the HsdM-like subunit. In type IIB RM enzymes,

DNA recognition is performed by an HsdS-like subunit
with two TRDs, but for the type IIG restriction enzyme
BpuSI, only one TRD with an inserted amino acid sequence is present. The type IIB RM enzyme is effectively
a dimer of a type IIG RM enzyme. Thus, a type IIB RM
enzyme is a motor-less type I RM system, and a type IIG
system is half of a motor-less type I RM enzyme. Figure 7
compares the relative locations of one endonuclease
domain, one HsdM, and the HsdS from the closed form
of EcoR124I with the structures of the type IIG enzymes
MmeI and BpuSI (Nakonieczna et al. 2009; Shen et al.
2011). MmeI recognizes the sequence TCCRAC and cuts
downstream at N20/N18 or N21/N19. BpuSI recognizes
the sequence GGGAC and cuts downstream at N10/N14.
It can be seen how fusion of the endonuclease domain from
HsdR to the start of HsdM in EcoR124I would move it to
the same location as observed in the type IIG restriction
enzymes and lead to cleavage downstream from the target
sequence. Thus, the recently proposed role of gene fusions
in the evolution of different groups of type II RM enzyme
systems (Mokrishcheva et al. 2011) can be extended to
include the evolution of the type I RM enzyme systems.
Materials and methods
Full details are given in the Supplemental Material.
Protein expression and purification
EcoR124I MTase and EcoR124I HsdR (Taylor et al. 1992; ObarskaKosinska et al. 2008), EcoKI (Dryden et al. 1997), and the Ocr anti-

restriction protein (Stephanou et al. 2009) were expressed and

purified as described previously.
Formation of protein and proteinDNA complexes
The R1M2S1 and R2M2S1 complexes of EcoR124I were formed by
incubation of purified HsdR and EcoR124I MTase at 1:1 and 1:2
molar ratios, respectively. Formation of the proteinDNA and
proteinOcr complexes used a 1:1.5 molar ratio of protein to
DNA or protein to Ocr dimer for EM studies and a 1:1 ratio of
protein to DNA for scattering studies. The oligonucleotides used
are described in the Supplemental Material.
EM and image processing
Negative stain grids were prepared by placing solutions of the
protein complexes (;40 mg/mL; i.e., ;100 nM) onto UV-treated
(Walker et al. 1985) continuous carbon-coated copper grids, then
stained with 1% uranyl acetate solution. EcoR124I+DNA samples
were prepared with a 30-bp fragment containing a centrally located
recognition sequence, while EcoKI+DNA samples were prepared
with a 75-bp fragment containing a centrally located recognition
sequence. Grids were viewed in a Jeol 1200EX electron microscope
fitted with a LaB6 (for EcoKI) or tungsten (for EcoR124I) electron
source operating at 80 kV. Negatives (Kodak SO63) were recorded
at 40,0003 magnification with defocus ranging from ;250 nm to
;850 nm and were digitized at 15 mm (EcoR124I) or 20 mm (EcoKI)
step size using an Imacon scanner. There were 3806 and 2647
negative stain particles in the EcoR124I6DNA data sets, respectively, and 2330 +Ocr particles. EcoKI negative stain data sets had
8300 unbound and 5910 +DNA particles.
EM maps have been submitted to Electron Microscopy Data
Bank (EMDB) with the following accession codes: EcoR124+DNA
1890: EcoR124I (no DNA) 1891: EcoR124I+Ocr 1892: EcoKI+DNA
1893.

Figure 7. Structural evolution of type IIG RM enzymes from a type I RM enzyme undergoing fusion of the C terminus of an
endonuclease domain from HsdR, via deletion of the motor domains, to the N terminus of HsdM. The structure on the left shows part
of EcoR124I, with one endonuclease domain from HsdR (in red), one HsdM (N-terminal domain is in green, and the MTase catalytic
domain is in blue), and the HsdS (in yellow) (two TRDs). DNA bound to the MTase core is shown, but DNA bound to HsdR is omitted
for clarity. The dashed line shows how the end of the endonuclease domain could join with the N terminus of HsdM to form a structure
similar to the type IIG structures shown on the right. The catalytic motifs in the endonuclease domain and HsdM are shown in
spacefill. The middle structure shows the structural model of MmeI with bound DNA with the same coloring used for equivalent
domains (endonuclease domain, N-terminal domain, MTase catalytic domain, and TRD) (Nakonieczna et al. 2009; coordinates from
ftp://genesilico.pl/iamb/models/RM.MmeI). The structure on the right shows the crystallographic structure of BpuSI (PDB code: 3s1s)
with the same coloring of domains as in the other structures and with an inserted extra domain shown in gray (Shen et al. 2011). DNA
is absent in this structure, and one can see that the endonuclease domain would be blocking the DNA-binding site on the TRD. Shen
et al. (2011) proposed that the endonuclease domain would twist away to allow DNA sequence recognition.

GENES & DEVELOPMENT

101

Kennaway et al.

SAXS
SAXS measurements were performed on a Bruker Nanostar
. For
instrument using Cu Ka radiation with a wavelength of 1.54 A
EcoKI, 16 1-h measurements were taken of the sample (0.85 mg/mL)
in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM Na2EDTA.
For EcoR124I, two 1-h measurements were taken of the sample (2.2
mg/mL) in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM
Na2EDTA. The sample-to-detector distance was 700 mm, providing
1. Samples were maintained at 10C.
a q-range of 0.080.32 A
No radiation damage was detected when successive data sets were
overlaid.
SANS
The HsdR of EcoR124I were deuterated by expression in E. coli
BL21 (DE3) cells using Enfors minimal medium containing 85%
D2O with hydrogenated glycerol as the carbon source. Endonuclease complexes were formed by adding HsdR to the MTase,
then dialyzing into 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and
1 mM Na2EDTA in varying H2O/D2O ratios. The complexes
were further characterized by dynamic light scattering (data not
shown). The protein concentration was between 2 and 4 mg/mL
in these experiments (510 mM endonuclease). SANS data were
collected using the D22 diffractometer at the ILL using two
detector distances, 2 and 10 m, for 15 and 30 min, respectively, at
1
10C, covering a scattering vector (q) range of 0.0080.35 A

with a wavelength of 6 A. A 96 3 96-cm detector with a pixel size

of 7.5 3 7.5 mm was used. Data for water were collected at a
detector distance of 4 m, allowing the data to be placed on an
absolute scale.
Construction of atomic models for type I RM complexes
Previous models of HsdS from EcoR124I (Obarska et al. 2006) and
EcoKI MTase (Kennaway et al. 2009) and crystal structures of
HsdM (3lkd) and HsdR (2w00) provided the starting points for
constructing atomic models of EcoR124I and EcoKI. The open
form of EcoR124I was constructed by independent fitting of two
copies of the R1M1 subcomplex and the S subunit taken from the
closed conformation. Model coordinates are available from us
and at ftp://genesilico.pl/iamb/models/RM.typeI.

Acknowledgments
We dedicate this paper to the memory of Professor Noreen
E. Murray (19352011). We thank Dr. Robert Knott for help in
performing small-angle X-ray measurements at the Bragg Institute,
ANSTO, Australia. D.T.F.D. would particularly like to acknowledge
many years of support and encouragement from Professors Noreen
and Kenneth Murray. We acknowledge funding from the Biotechnology and Biological Sciences Research Council (BB/D001870/1 to
J.T. and D.T.F.D.), the Wellcome Trust (080304/Z/06/Z to G.G.K.),
and the Foundation for Polish Science (TEAM/2009-4/2 to J.M.B.).
The EM work on EcoKI was initiated as a result of the Isaac
Newton Institute for Mathematical Sciences Workshop on
Statistical Mechanics of Molecular and Cellular Biological
Systems, JanuaryJuly 2004.

References
Abadjieva A, Patel J, Webb M, Zinkevich V, Firman K. 1993. A
deletion mutant of the type IC restriction endonuclease
EcoR1241 expressing a novel DNA specificity. Nucleic Acids
Res 21: 44354443.
Atanasiu C, Su TJ, Sturrock SS, Dryden DTF. 2002. Interaction
of the Ocr gene 0.3 protein of bacteriophage T7 with EcoKI

102

GENES & DEVELOPMENT

restriction/modification enzyme. Nucleic Acids Res 30:

39363944.
Calisto BM, Pich OQ, Pinol J, Fita I, Querol E, Carpena X. 2005.
Crystal structure of a putative type I restriction-modification S
subunit from Mycoplasma genitalium. J Mol Biol 351: 749762.
Callow P, Sukhodub A, Taylor J, Kneale G. 2007. Shape and
subunit organisation of the DNA methyltransferase M.AhdI.
J Mol Biol 369: 177185.
Chen K, Roberts GA, Stephanou AS, Cooper LP, White JH, Dryden
DTF. 2010. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of type I DNA restriction and
modification enzymes. Biochem Biophys Res Commun 398:
254259.
Cooper LP, Dryden DTF. 1994. The domains of a type I DNA
methyltransferase Interactions and role in recognition of
DNA methylation. J Mol Biol 236: 10111021.
Cowan GM, Gann AA, Murray NE. 1989. Conservation of
complex DNA recognition domains between families of
restriction enzymes. Cell 56: 103109.
Davies GP, Martin I, Sturrock SS, Cronshaw A, Murray NE, Dryden
DTF. 1999. On the structure and operation of type I DNA
restriction enzymes. J Mol Biol 290: 565579.
Dryden DTF. 1999. Bacterial DNA methyltransferases. In
S-adenosylmethionine-dependent methyltransferases: Structures and functions (ed. X Cheng, RM Blumenthal), pp. 283
340. World Scientific Publishing, Singapore.
Dryden DTF, Sturrock SS, Winter M. 1995. Structural modelling of
a type I DNA methyltransferase. Nat Struct Biol 2: 632635.
Dryden DTF, Cooper LP, Thorpe PH, Byron O. 1997. The in vitro
assembly of the EcoKI type I DNA restriction/modification
enzyme and its in vivo implications. Biochemistry 36: 1065
1076.
Durr H, Korner C, Muller M, Hickmann V, Hopfner KP. 2005.
X-ray structures of the Sulfolobus solfataricus SWI2/SNF2
ATPase core and its complex with DNA. Cell 121: 363373.
Endlich B, Linn S. 1985. The DNA restriction endonuclease of
Escherichia coli B. I. Studies of the DNA translocation and
the ATPase activities. J Biol Chem 260: 57205728.
Fuller-Pace FV, Bullas LR, Delius H, Murray NE. 1984. Genetic
recombination can generate altered restriction specificity.
Proc Natl Acad Sci 81: 60956099.
Gann AA, Campbell AJB, Collins JF, Coulson AF, Murray NE.
1987. Reassortment of DNA recognition domains and the
evolution of new specificities. Mol Microbiol 1: 1322.
Garca LR, Molineux IJ. 1999. Translocation and specific cleavage of bacteriophage T7 DNA in vivo by EcoKI. Proc Natl
Acad Sci 96: 1243012435.
Gao P, Tang Q, An X, Yan X, Liang D. 2011. Structure of HsdS
subunit from Thermoanaerobacter tengcongensis sheds
lights on mechanism of dynamic opening and closing of
type I methyltransferase. PLoS ONE 6: e17346. doi: 101371/
journalpone0017346.
Griffith JD. 1978. DNA structure: Evidence from electron microscopy. Science 201: 525527.
Gubler M, Bickle TA. 1991. Increased protein flexibility leads to
promiscuous proteinDNA interactions in type IC restriction-modification systems. EMBO J 10: 951957.
Janscak P, Dryden DTF, Firman K. 1998. Analysis of the subunit
assembly of the type IC restrictionmodification enzyme
EcoR124I. Nucleic Acids Res 26: 44394445.
Kannan P, Cowan GM, Daniel AS, Gann AA, Murray NE. 1989.
Conservation of organization in the specificity polypeptides
of two families of type I restriction enzymes. J Mol Biol 209:
335344.
Kennaway CK, Obarska-Kosinska A, White JH, Tuszynska I,
Cooper LP, Bujnicki JM, Trinick J, Dryden DTF. 2009. The

Structure of type I restriction enzymes

structure of M.EcoKI type I DNA methyltransferase with

a DNA mimic antirestriction protein. Nucleic Acids Res 37:
762770.
Kim JS, De Giovanni A, Jancarik J, Adams PD, Yokota H, Kim R,
Kim SH. 2005. Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and
its functional implications. Proc Natl Acad Sci 102: 3248
3253.
Kneale GG. 1994. A symmetrical model for the domain structure of type I DNA methyltransferases. J Mol Biol 243: 15.
Lapkouski M, Panjikar S, Janscak P, Smatanova IK, Carey J,
Ettrich R, Csefalvay E. 2009. Structure of the motor subunit
of type I restrictionmodification complex EcoR124I. Nat
Struct Mol Biol 16: 9495.
Linn S, Arber W. 1968. Host specificity of DNA produced by
Escherichia coli. X. In vitro restriction of phage fd replicative
form. Proc Natl Acad Sci 59: 13001306.
Loenen WA. 2003. Tracking EcoKI and DNA fifty years on: A
golden story full of surprises. Nucleic Acids Res 31: 7059
7069.
Madhusoodanan UK, Rao DN. 2010. Diversity of DNA methyltransferases that recognize asymmetric target sequences.
Crit Rev Biochem Mol Biol 45: 125145.
Makovets S, Powell LM, Titheradge AJB, Blakely GW, Murray
NE. 2004. Is modification sufficient to protect a bacterial
chromosome from a resident restriction endonuclease? Mol
Microbiol 51: 135147.
Mayanagi K, Kiyonari S, Nishida H, Saito M, Kohda D, Ishino Y,
Shirai T, Morikawa K. 2011. Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)DNA
ternary complex. Proc Natl Acad Sci 108: 18451849.
McClelland SE, Dryden DTF, Szczelkun MD. 2005. Continuous
assays for DNA translocation using fluorescent triplex
dissociation: Application to type I restriction endonucleases.
J Mol Biol 348: 895915.
McMahon SA, Roberts GA, Johnson KA, Cooper LP, Liu H,
White JH, Carter LG, Sanghvi B, Oke M, Walkinshaw MD,
et al. 2009. Extensive DNA mimicry by the ArdA antirestriction protein and its role in the spread of antibiotic
resistance. Nucleic Acids Res 37: 48874897.
Meister J, MacWilliams M, Hubner P, Jutte H, Skrzypek E,
Piekarowicz A, Bickle TA. 1993. Macroevolution by transposition: Drastic modification of DNA recognition by a type
I restriction enzyme following Tn5 transposition. EMBO J
12: 45854591.
Melero R, Rajagopalan S, Lazaro M, Joerger AC, Brandt T,
Veprintsev DB, Lasso G, Gil D, Scheres SH, Carazo JM,
et al. 2011. Electron microscopy studies on the quaternary
structure of p53 reveal different binding modes for p53
tetramers in complex with DNA. Proc Natl Acad Sci 108:
557562.
Mernagh DR, Janscak P, Firman K, Kneale GG. 1998. Protein
protein and proteinDNA interactions in the type I restriction endonuclease R.EcoR124I. Biol Chem 379: 497503.
Meselson M, Yuan R. 1968. DNA restriction enzyme from E.
coli. Nature 217: 11101114.
Mokrishcheva ML, Solonin AS, Nikitin DV. 2011. Fused eco29kIRand M genes coding for a fully functional hybrid polypeptide
as a model of molecular evolution of restrictionmodification
systems. BMC Evol Biol 11: 35.
Murray NE. 2000. Type I restriction systems: Sophisticated
molecular machines (a legacy of Bertani and Weigle). Microbiol Mol Biol Rev 64: 412434.
Murray NE. 2002. 2001 Fred Griffith review lecture Immigration
control of DNA in bacteria: Self versus non-self. Microbiology
148: 320.

Nakonieczna J, Kaczorowski T, Obarska-Kosinska A, Bujnicki JM.

2009. Functional analysis of MmeI from methanol utilizer
Methylophilus methylotrophus, a subtype IIC restriction
modification enzyme related to type I enzymes. Appl Environ
Microbiol 75: 212223.
Neaves KJ, Cooper LP, White JH, Carnally SM, Dryden DTF,
Edwardson JM, Henderson RM. 2009. Atomic force microscopy of the EcoKI type I DNA restriction enzyme bound to
DNA shows enzyme dimerization and DNA looping.
Nucleic Acids Res 37: 20532063.
Nekrasov SV, Agafonova OV, Belogurova NG, Delver EP, Belogurov
AA. 2007. Plasmid-encoded antirestriction protein ArdA
can discriminate between type I methyltransferase and
complete restrictionmodification system. J Mol Biol 365:
284297.
Obarska A, Blundell A, Feder M, Vejsadova S, Sisakova E,
Weiserova M, Bujnicki JM, Firman K. 2006. Structural model
for the multisubunit type IC restriction-modification DNA
methyltransferase MEcoR124I in complex with DNA. Nucleic
Acids Res 34: 19922005.
Obarska-Kosinska A, Taylor JEA, Callow P, Orlowski J, Bujnicki
JM, Kneale GG. 2008. HsdR subunit of the type I restrictionmodification enzyme EcoR124I: Biophysical characterisation
and structural modelling. J Mol Biol 376: 438452.
Powell LM, Dryden DTF, Murray NE. 1998. Sequence-specific
DNA binding by EcoKI a type IA DNA restriction enzyme.
J Mol Biol 283: 963976.
Powell LM, Lejeune E, Hussain FS, Cronshaw AD, Kelly SM,
Price NC, Dryden DTF. 2003. Assembly of EcoKI DNA
methyltransferase requires the C-terminal region of the
HsdM modification subunit. Biophys Chem 103: 129
137.
Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA,
Bitinaite J, Blumenthal RM, Degtyarev SKh, Dryden DTF,
Dybvig K, et al. 2003. A nomenclature for restriction enzymes,
DNA methyltransferases, homing endonucleases and their
genes. Nucleic Acids Res 31: 18051812.
Roberts RJ, Vincze T, Posfai J, Macelis D. 2010. REBASEa
database for DNA restriction and modification: Enzymes
genes and genomes. Nucleic Acids Res 38: D234D236. doi:
10.1093/nar/gkp874.
Roberts GA, Cooper LP, White JH, Su TJ, Zipprich JT, Geary P,
Kennedy C, Dryden DTF. 2011. An investigation of the
structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI type I DNA restriction and modification
enzyme. Nucleic Acids Res 39: 76677676.
Seidel R, van Noort J, van der Scheer C, Bloom JGP, Dekker NH,
Dutta CF, Blundell A, Robinson T, Firman K, Dekker C.
2004. Real-time observation of DNA translocation by the
type I restriction modification enzyme EcoR124I. Nat Struct
Mol Biol 11: 838843.
Seidel R, Bloom JG, Dekker C, Szczelkun MD. 2008. Motor step
size and ATP coupling efficiency of the dsDNA translocase.
EcoR124I. EMBO J 27: 13881398.
Shen BW, Xu D, Chan SH, Zheng Y, Zhu Z, Xu SY, Stoddard BL.
2011. Characterization and crystal structure of the type IIG
restriction endonuclease RM.BpuSI. Nucleic Acids Res 39:
82238236.
Simons M, Szczelkun MD. 2011. Recycling of protein subunits
during DNA translocation and cleavage by type I restrictionmodification enzymes. Nucleic Acids Res 39: 76567666.
Stephanou AS, Roberts GA, Tock MR, Pritchard EH, Turkington
R, Nutley M, Cooper A, Dryden DTF. 2009. A mutational
analysis of DNA mimicry by Ocr the gene 0.3 antirestriction
protein of bacteriophage T7. Biochem Biophys Res Commun
378: 129132.

GENES & DEVELOPMENT

103

Kennaway et al.

Studier FW, Bandyopadhyay PK. 1988. Model for how type I

restriction enzymes select cleavage sites in DNA. Proc Natl
Acad Sci 85: 46774681.
Svergun DI. 1999. Restoring low resolution structure of biological macromolecules from solution scattering using
simulated annealing. Biophys J 76: 28792886.
Svergun DI, Nierhaus KH. 2000. A map of proteinrRNA
distribution in the 70 S Escherichia coli ribosome. J Biol
Chem 275: 1443214439.
Taylor IA, Patel J, Firman K, Kneale GG. 1992. Purification and
biochemical-characterization of the EcoR124 type-I modification methylase. Nucleic Acids Res 20: 179186.
Taylor IA, Davis KG, Watts D, Kneale GG. 1994. DNA-binding
induces a major structural transition in a type-I methyltransferase. EMBO J 13: 57725778.
Taylor JE, Callow P, Swiderska A, Kneale GG. 2010. Structural
and functional analysis of the engineered type I DNA
methyltransferase EcoR124INT. J Mol Biol 398: 391399.
Tock MR, Dryden DTF. 2005. The biology of restriction and
anti-restriction. Curr Opin Microbiol 8: 466472.
Uyen NT, Park S, Choi J, Lee HJ, Nishi K, Kim JS. 2009. The
fragment structure of a putative HsdR subunit of a type I
restriction enzyme from Vibrio vulnificus YJ016: Implications for DNA restriction and translocation activity. Nucleic
Acids Res 37: 69606969.
van Noort J, van der Heijden T, Dutta CF, Firman K, Dekker C.
2004. Initiation of translocation by type I restriction-modification enzymes is associated with a short DNA extrusion.
Nucleic Acids Res 32: 65406547.
Waldron DE, Lindsay JA. 2006. Sau1: A novel lineage-specific
type I restriction-modification system that blocks horizontal
gene transfer into Staphylococcus aureus and between S.
aureus isolates of different lineages. J Bacteriol 188: 5578
5585.
Walker M, Knight P, Trinick J. 1985. Negative staining of
myosin molecules. J Mol Biol 184: 535542.
Walkinshaw MD, Taylor P, Sturrock SS, Atanasiu C, Berge T,
Henderson RM, Edwardson JM, Dryden DTF. 2002. Structure
of Ocr from bacteriophage T7 a protein that mimics B-form
DNA. Mol Cell 9: 187194.
Webb M, Taylor IA, Firman K, Kneale GG. 1995. Probing the
domain structure of the type IC DNA methyltransferase
M.EcoR124I by limited proteolysis. J Mol Biol 250: 181190.
Yuan R, Hamilton DL, Burckhardt J. 1980. DNA translocation
by the restriction enzyme from E. coli K. Cell 20: 237244.

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GENES & DEVELOPMENT